Project Summary Mycobacterium tuberculosis (Mtb) infects the lungs causing 1.6 million deaths a year. The only vaccine approved for tuberculosis has limited efficacy. Additionally, antibiotic treatment requires six months, but can require two or more years for multi-drug resistant strains that are becoming more prevalent. Therefore, development of a host-directed therapeutic strategy that directly targets the Mtb reservoir would dramatically impact human health. I propose to identify novel innate immune factors that protect against Mtb infection in vivo, which could be the basis of such a therapeutic approach. Specific Aims: During Mtb infection, inflammatory cytokines like IL-1 stimulate non-infected cells to produce factors that enhance anti-bacterial activity of Mtb-bearing cells. While IL-1 is critical for protection against Mtb, its mechanism of action is unknown. Aim 1) We have identified IL-1 sensing cells in Mtb-infected lungs and IL-1-induced genes expressed by these cells. I will test whether natural killer T 17 cells, one of the IL-1 sensing populations, provides protection against Mtb. Additionally, I will determine which IL-1-induced genes act directly on Mtb infected macrophages in vivo to confer the protective effect of IL-1. Aim 2) Neutrophils are detrimental in Mtb- susceptible mouse strains and neutrophilia positively correlates with bacterial burden and mortality in humans. However, it is unclear how neutrophils interfere with the host response to Mtb. We have previously shown that IL-1 receptor antagonist (IL-1Ra) is an immunosuppressive molecule that limits Mtb control in a susceptible mouse strain. As IL-1Ra is highly expressed by neutrophils, I will determine whether neutrophils inhibit the protective response against Mtb through their production of IL-1Ra and whether this immunosuppressive function is conserved across multiple Mtb susceptible strains. Additionally, I will determine whether neutrophils exploit other immunosuppressive pathways, like IL-18 signaling inhibition and PD-1, to hinder the productive immune response to Mtb. Study Design: I will define how communication between non-infected and infected cells leads to control of tuberculosis infection by generating mixed bone marrow chimeras. These experiments will test the in vivo relevance of IL-1-driven gene expression for candidates identified in our single cell RNA- sequencing dataset of Mtb-infected lungs. Additionally, I will leverage our single cell RNA-sequencing dataset to determine how neutrophils inhibit control of the infection in Mtb-sensitive mouse strains. Potential Impact: The identification of protective and inhibitory regulators of Mtb control can serve as the basis for host-directed therapies to treat Mtb infection, which will be a major advance for the field.